通过DNA改组技术定向进化赖氨酸脱羧酶基因cadA和ldc

Directed evolution by DNA shuffling of lysine decarboxylase gene cad A and ldc

利用DNA改组技术对赖氨酸脱羧酶野生型基因ldc进行随机突变,在大肠杆菌Escherichia coli JM109中构建赖氨酸脱羧酶突变体库。从E.coli JM109和蜂房哈夫尼菌Hafnia alvei AS1.1009中分别克隆出赖氨酸脱羧酶基因cad A和ldc。查询NCBI数据库得知,二者的同源性为75%。分别构建重组质粒p Trc99a-cad A和p Trc99a-ldc,以此2种质粒为模板,经PCR扩增,获得目的基因片段,分析目的基因片段中存在的限制性酶切位点,用多种限制性内切酶碎片化2种基因,切割成不同大小的片段。这些小片段进行不同组合,突变体经过LBXL平板初筛和高效液相色谱(HPLC)复筛,获得1株酶活性提高的赖氨酸脱羧酶突变体,编号为LDC2-16,其比酶活为4 869.86 U/mg(以1 mg总蛋白计),与2种野生型赖氨酸脱羧酶基因表达的酶Cad A(1 652.63 U/mg)、Ldc(2 365.93 U/mg)相比,在最适温度37℃、p H 6.0时,突变体的比酶活分别是上述野生型酶的2.95和2.06倍。摇瓶发酵5 h后,目标产物1,5-戊二胺产量从46.9提高至63.9 g/L,提高了36%。

We enhanced the activity of two lysine decarboxylase（LDC） cadA and ldc by DNA shuffling through random mutation. One lysine decarboxylase gene cadA from Escherichia coli and the other ldc from Hafnia alvei AS1.1009 were linked into vector pTrc99a. Referring to NCBI database,the nucleotide sequence analysis showed 75% homology between cadA gene and ldc gene. Then, we obtained three recombinant plasmid pTrc99a？cadA and pTrc99a？ldc. As templates, two plasmids were amplified by normal PCR to obtain the genes cadA and ldc. According to the restriction enzyme sites present in the two genes,we used variety of restriction enzyme sites to get endonuclease fragments. Then, the random fragments were purified and reassembled by PCR without primers and the products were amplified by routine PCR. These fragments were successfully reassembled to genes of the same size as LDC. The activity of LDC2？16 can reach to 4 869？86 U/mg total protein,which is 2？95 and 2？06 times as that of the wide type （ CadA 1 652？63 U/mg、 Ldc 2 365？93 U/mg ） under the optimum conditions ofnbsp;temperature 37 ℃, pH 6？0. After 5 h fermentation, the target product 1, 5？diaminopentane production researched 63？9 g/L from 46？9 g/L with an increase of 36%.